Interactions of substrates and alpha-lactalbumin with galactosyltransferase as measured by difference spectroscopy.
The interactions of Mn2+, nucleotides, monosaccharide substrates, and the modifier protein alpha-lactalbumin with galactosyltransferase have been studied by difference spectroscopy. MnCl2, required for significant binding of UDP-galactose or UDP to the enzyme, does not exhibit specific interactions with tyrosine or tryptophan residues. The interaction of UDP-galactose or UDP with galactosyltransferase in 2 mM MnCl2 produces difference spectra with a major positive peak at 284 nm, a second positive peak at 298 nm, and a large negative trough at 254 nm, suggesting the involvement of tyrosine and tryptophan residues in the interaction. The interaction of GlcNAc with the enzyme in 2 mM MnCl2 produces only small nonspecific difference spectra. However, the addition of 100 mM GlcNAc markedly increases the difference extinction coefficient at 284 nm of the UDP-bound enzyme-ligand complex, while the coefficient at 254 nm which arises from UDP remains constant. The results suggest that a conformational change involving tyrosine residues, which does not affect UDP, occurs in the process, enzyme.Mn + UDP + GlcNAc leads to enzyme.Mn.UDP.GlcNAc. Glucose does not show a similar effect. The interaction of galactosyltransferase and alpha-lactalbumin produces difference spectra characteristic of tryptophan and does not affect the difference spectra of galactosyltransferase produced by the interaction with UDP and GlcNAc. This implies that the interaction of the two proteins does not involve the bound UDP on galactosyltransferase and does not affect the conformational change induced by UDP and GlcNAc.
D000818:Animals; D002417:Cattle; D005260:Female; D005700:Galactosyltransferases / Q000378:metabolism*; D007700:Kinetics; D007768:Lactalbumin / Q000378:metabolism*; D008024:Ligands; D008345:Manganese / Q000494:pharmacology; D008892:Milk / Q000201:enzymology; D013056:Spectrophotometry, Ultraviolet
D008024:Ligands; D008345:Manganese; D007768:Lactalbumin; D005700:Galactosyltransferases